School outbreak of Escherichia coli O157 with high levels of transmission, Staffordshire, England, February 2012.

BACKGROUND: Verocytotoxin-producing Escherichia coli (VTEC) are bacteria that cause infectious gastroenteritis and in certain settings can cause widespread infection due to secondary transmission. We describe the findings of an investigation of a school-based outbreak of VTEC in Staffordshire, England. METHODS: Outbreak investigation at a school in February 2012 after two children were diagnosed with VTEC infection. Cases were defined as pupils and staff (or their household contacts) with gastrointestinal symptoms or asymptomatic screened persons, with laboratory confirmed VTEC O157 infection (phage type 32, verocytotoxin 2) occurring on or after 1 February 2012. Microbiological tests of food and faecal samples plus screening of asymptomatic contacts were undertaken. Epidemiological and clinical data were descriptively analysed. RESULTS: Thirty-eight cases were detected. Nineteen were asymptomatic and identified via screening of 191 pupils. Infection was introduced into the school from an earlier household cluster, followed by extensive person-to-person transmission within the nursery/infant group with limited spread to the wider school population. CONCLUSIONS: Control measures included several interventions, in particular, universal screening of pupils and staff. Screening during school outbreaks is not underpinned by guidance but proved to be a key control measure. Screening of asymptomatic contacts should be considered in similar outbreaks.

Rapid draft sequencing and real-time nanopore sequencing in a hospital outbreak of Salmonella.

BACKGROUND: Foodborne outbreaks of Salmonella remain a pressing public health concern. We recently detected a large outbreak of Salmonella enterica serovar Enteritidis phage type 14b affecting more than 30 patients in our hospital. This outbreak was linked to community, national and European-wide cases. Hospital patients with Salmonella are at high risk, and require a rapid response. We initially investigated this outbreak by whole-genome sequencing using a novel rapid protocol on the Illumina MiSeq; we then integrated these data with whole-genome data from surveillance sequencing, thereby placing the outbreak in a national context. Additionally, we investigated the potential of a newly released sequencing technology, the MinION from Oxford Nanopore Technologies, in the management of a hospital outbreak of Salmonella. RESULTS: We demonstrate that rapid MiSeq sequencing can reduce the time to answer compared to the standard sequencing protocol with no impact on the results. We show, for the first time, that the MinION can acquire clinically relevant information in real time and within minutes of a DNA library being loaded. MinION sequencing permits confident assignment to species level within 20 min. Using a novel streaming phylogenetic placement method samples can be assigned to a serotype in 40 min and determined to be part of the outbreak in less than 2 h. CONCLUSIONS: Both approaches yielded reliable and actionable clinical information on the Salmonella outbreak in less than half a day. The rapid availability of such information may facilitate more informed epidemiological investigations and influence infection control practices.

Rapid genotyping of CTX-M extended-spectrum beta-lactamases by denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (dHPLC) is a powerful technique which has been used extensively to detect genetic variation. This is the first report of the application of dHPLC for rapid genotyping of bacterial beta-lactamase genes. The technique was specifically developed to genotype members of all blaCTX-M DNA homology groups. Thirteen well-defined blaCTX-M extended-spectrum beta-lactamase (ESBL)-producing strains were used to develop and optimize the dHPLC genotyping assay. Further evaluation was carried out with a blinded panel of 62 clinical isolates. The results of blaCTX-M genotyping achieved by dHPLC were comparable to the typing results obtained by DNA sequencing. Applying the newly developed dHPLC-based genotyping method, we successfully genotyped all 73 blaCTX-M ESBL-producing strains from the 4-month survey study. Furthermore, we found the first reported cases in the United Kingdom of clinically significant disease caused by CTX-M-14- and CTX-M-1-producing Escherichia coli strains. We conclude that the novel dHPLC assay is highly accurate, rapid, and cost-effective for the genotyping of blaCTX-M-producing ESBLs and has great potential for determining the clinical relevance of different and new blaCTX-M genotypes, as well as for epidemiological studies and surveillance programs.

"Random" gentamicin concentrations do not predict trough levels in neonates receiving once daily fixed dose regimens.

BACKGROUND: Monitoring plasma gentamicin concentrations in neonates 24 hours after a once daily dose (4 mg/kg) often necessitates additional blood sampling. In adults a nomogram has been developed enabling evaluation of gentamicin doses by sampling concentrations with other blood tests, 4-16 hours after administration. We attempted to develop a similar nomogram for neonates. METHODS: In addition to standard 24 hour sampling to monitor trough concentrations, one additional "random" gentamicin concentration was measured in each of 50 neonates < 4 days of age (median gestation 33 weeks [28-41]), when other blood samples were clinically necessary, 4-20 hours after gentamicin administration. 24 hour concentrations of > 1 mg/L were considered high, and an indication to extend the dosing interval. RESULTS: Highest correlation (r2 = 0.51) of plasma gentamicin concentration against time (4 to 20 hours) was with logarithmic regression. A line drawn 0.5 mg/L below the true regression line resulted in all babies with 24 hr gentamicin concentrations > 1 mg/L having the additional "random" test result above that line, i.e. 100% sensitivity for 24 hour concentrations > 1 mg/L, though only 58% specificity. Having created the nomogram, 39 further babies (median gestation 34 weeks [28-41]), were studied and results tested against the nomogram. In this validation group, sensitivity of the nomogram for 24 hr concentrations > 1 mg/L was 92%; specificity 14%, positive predictive value 66%, and negative predictive value 50%. Prematurity (< or = 37 weeks) was a more sensitive (94%) and specific (61%) indicator of high 24-hour concentrations. 62 (87%) of 71 preterm babies had high 24-hour concentrations. CONCLUSION: It was not possible to construct a nomogram to predict gentamicin concentrations at 24 hours in neonates with a variety of gestational ages. Dosage tailored to gestation with monitoring of trough concentrations remains management of choice.

Rapid and simple detection of blaCTX-M genes by multiplex PCR assay.

A novel multiplex PCR assay is described (CTX-Mplex PCR) that allows rapid detection of bla(CTX-M) genes and discrimination between groups 1, 2, 9 and 25/26. The specificity and sensitivity of the assay were evaluated with 10 control strains and then applied to 62 clinical isolates. The multiplex PCR detected and classified bla(CTX-M) genes with 100 % accuracy. The utilization of a denaturing HPLC WAVE system to size the PCR products automatically from the multiplex PCR enhances the assay by saving time and costs.